Velocity factor

ABSTRACT

The current invention is directed to the velocity factor. Based on the velocity factor antibodies can be classified, i.e. antibodies can be characterized on their binding properties as e.g. entropic or enthalpic antigen binder. A velocity factor based classification does not require detailed thermodynamic determinations and/or calculations. The velocity factor is the ratio of the antigen-antibody complex association rate constants ka determined at 37° C. and 13° C. As only two experimental determinations are required to calculate the velocity factor this is a fast and high-throughput suited method.

This application is a divisional of U.S. application Ser. No. 13/392,217filed Feb. 24, 2012, which is now U.S. Pat. No. 8,617,849, which is anational stage entry of International Application No. PCT/EP2010/062115,filed Aug. 19, 2010, which claims the benefit of European PatentApplication No. EP 09010844.0, filed Aug. 25, 2009. The contents of eachof these Applications are hereby incorporated by reference in theirentirety.

The velocity factor is the ratio of the antigen-antibody complexassociation rate constants determined at 37° C. and 13° C. with which itis possible to classify antibodies e.g. to be an entropic or enthalpicbinder without the need of detailed thermodynamic determinations andcalculations.

BACKGROUND OF THE INVENTION

The generation of high-affinity antibodies with distinguished antigenspecificity and extraordinary antigen complex stability is a major goalin diagnostic and therapeutic antibody development.

Regarding thermodynamic analyses of protein-protein interactions theprevailing technology is the calorimetric assay (Chaires, J. B., Ann.Rev. Biophys. 37 (2008) 135-51; Perozzo, R., et al., J. Recept. SignalTransduct. Res. 24 (1-2) (2004) 1-52; Li, L., et al., Chem. Biol. DrugDes. 71(6) (2008) 529-32; Liang, Y., Acta Biochim. Biophys. Sin.(Shanghai) 40(7) (2008) 565-76; Thielges, M. C., et al., Biochemistry47(27) (2008) 7237-47). The required sample amount for a reactioncalorimeter determination is high, such as an antibody concentration ofat least 125 μg/ml and sample volumes of at least 150 μl. Furthermorereaction calorimetry requests high sample purity and does not tolerateany sample impurities or sample heterogeneity. Additionally the samplebuffers directly influence the results of the determined thermodynamicparameters. Further, reaction calorimetry is solely able to resolveequilibrium thermodynamics.

Surface Plasmon Resonance (SPR) instrumentation (Roos, H., et al., J.Mol. Recognit. 11(1-6) (1998) 204-210; Van Regenmortel, M. H., et al.,J. Mol. Recognit. 11(1-6) (1998) 163-167; Gunnarsson, K., Curr. Prot.Immunol. Chapter 18 (2001) Unit 18.6; Drake, A. W., et al., Anal.Biochem. 328(1) (2004) 35-43; Kikuchi, Y., et al., J. Biosci. Bioeng.100(3) (2005) 311-7) allows the rapid determination oftemperature-dependent kinetic profiles in a high throughput manner (seee.g. Canziani, G. A., et al., Anal. Biochem. 325 (2004) 301-307;Saefsten, P., et al., Anal. Biochem. 353 (2006) 181-190; Leonard, P., etal., J. Immunol. Methods 323 (2007) 172-9).

Wassaf, D., et al. (Anal. Biochem. 351 (2006) 241-253) reporthigh-throughput affinity ranking of antibodies using surface plasmonresonance microarrays. A thermodynamic analysis of protein interactionswith biosensor technology is reported in Roos, H., et al., J. Mol.Recognit. 11 (1998) 204-210.

Wark, K. L., et al. (Adv. Drug. Deliv. Rev. 58 (2006) 657-670) reporttechnologies for the enhancement of antibody affinity. High-resolutioncharacterization of antibody fragment/antigen interactions using BIAcoreT100 is reported by Papalia, G. A., et al. in Anal. Biochem. 359 (2006)112-119. Sagawa, T., et al. (Mol. Immunol. 39 (2003) 801-808) reportthermodynamic and kinetic aspects of antibody evolution during theimmune response to hapten. An overview of BIAcore systems andapplication is reported by Jason-Moller, L., et al. in Curr. Prot. Prot.Sci. (2006) page unit 19.13. Gelb, W., reports microcalorimetric methodsfor peptides studies (Proc. 4th Int. Peptide Sympos. (2007) page 1).

SUMMARY OF THE INVENTION

It has been found that for the characterization of antibodies nothroughout kinetic characterization is required but furthermore thedetermination of the association rate constant at 37° C. and theassociation rate constant at 13° C. is required. The ratio thereof canbe used to classify antibodies according to their binding properties.

The first aspect as reported herein is a method for selecting anantibody comprising the step of selecting an antibody with a velocityfactor of 10 or less.

In one embodiment the method for selecting an antibody comprises thefollowing steps

-   -   a) providing a set of or a population or a multitude of        antibodies,    -   b) determining temperature-dependent kinetic data,    -   c) calculating the velocity factor for all provided antibodies,    -   d) selecting an antibody with a velocity factor of 10 or less.

In one embodiment the velocity factor is of from 0 to 10. In anotherembodiment the velocity factor is the ratio of the association rateconstant at 37° C. to the association rate constant at 13° C. In stillanother embodiment the temperature-dependent kinetic data is determinedat 13° C. and 37° C. In another embodiment the determiningtemperature-dependent kinetic data is with surface plasmon resonance. Ina further embodiment the method is a high-throughput method. In stillanother embodiment the selecting is by the velocity factor and by theΔS^(∘)‡ass value. In one embodiment in the surface plasmon resonance theantigen is immobilized on the surface plasmon resonance chip. In afurther embodiment the temperature-dependent kinetic data isthermodynamic equilibrium data and transition state thermodynamic data.In still another embodiment the temperature-dependent kinetic data iscalculated with the linear forms of the van't Hoff and Eyring andArrhenius equations. In one embodiment an antibody is selected with aΔS^(∘)‡ass of less than 200 J/mol*K (ΔS^(∘)‡ass<200 J/mol*K). In oneembodiment an antibody is selected with a ΔS^(∘)‡ass in the range offrom −200 J/mol*K to 200 J/mol*K. In also an embodiment an antibody isselected with a ΔS^(∘)‡ass in the range of from −150 J/mol*K to +100J/mol*K. In another embodiment the surface plasmon resonance is with theHBS-EP buffer supplemented with 1 mg/ml carboxymethyldextrane. In stillanother embodiment each of the antibodies is produced by a singlehybridoma or B-cell.

A second aspect as reported herein is a method for obtaining an antibodywith cross-reactivity for at least two antigens comprising selecting anantibody with a velocity factor of 50 or more. In one embodiment thevelocity factor is of from 50 to 300.

A further aspect as reported herein is a method for producing anantibody comprising the following steps:

-   -   a) providing a set/population/multitude of antibody producing        cells,    -   b) selecting an antibody producing cell producing an antibody        with a velocity factor of 10 or less,    -   c) cultivating the selected cell,    -   d) recovering the antibody from the cultivated selected cell or        the cultivation medium thereof and thereby producing an        antibody.

In one embodiment the antibody producing cells are deposited as singlecells. In a further embodiment the deposited single cells are cultivatedprior to the selecting. In another embodiment the antibody is purifiedprior to the selecting. In a further embodiment the antibody is purifiedafter the recovering. In a specific embodiment the purifying is byProtein A chromatography.

Another aspect as reported herein is a method for selecting a humanizedform of a parent antibody comprising

-   -   a) providing a parent antibody,    -   b) providing a set/population/multitude of humanized forms of        the parent antibody,    -   c) determining temperature-dependent kinetic data of all        provided antibodies,    -   d) calculating the velocity factor for all provided antibodies,    -   e) comparing the velocity factors of the humanized forms with        the velocity factor of the parent antibody,    -   f) selecting a humanized form of a parent antibody by selecting        an antibody with a velocity factor that is less than twice of        the velocity factor of the parent antibody.

In one embodiment the selecting is of a humanized from with a velocityfactor that is less than 125% of the velocity factor of the parentantibody, in another embodiment less than 110% of the velocity factor ofthe parent antibody.

Still another aspect as reported herein is the use of an antibodyselected with a method as reported herein as therapeutic or diagnosticagent. One aspect of the invention is a pharmaceutical compositioncomprising an antibody obtained with a method as reported herein.

DETAILED DESCRIPTION OF THE INVENTION

The methods reported herein are based on the determination of thevelocity factor. Based on the velocity factor antibodies can beclassified, i.e. antibodies can be characterized according to theirbinding properties as e.g. entropic or enthalpic antigen binder. It hasbeen found that a velocity factor based classification does not requiredetailed thermodynamic to be determined and/or calculated, thus,reducing the amount of parameters to be determined and the number ofcalculations to be performed. The velocity factor is the ratio of theantigen-antibody complex association rate constants (ka) independentlydetermined at 37° C. and at 13° C. As only two experimentaldeterminations are required to calculate the velocity factor this is afast and high-throughput suited method.

The velocity factor can be used e.g. to compare a humanized antibodywith the parent antibody from which it is derived. It can also be usedto evaluate antibodies directly from hybridoma and/or B-cellsupernatants. This does not require a preliminary purification as thedetermination can be made directly with the crude supernatant.

Surface plasmon resonance based kinetic methods have several advantagesover conventional calorimetric assays:

-   -   high throughput processing is possible,    -   low sample consumption,    -   measurement of affinity instead of avidity, and    -   use of crude cell supernatants or complex cultivation mixtures.

The surface plasmon biosensor surface is an affinity matrix, which isused for example for antibody capturing from crude cell cultivationsupernatants. Thus, crude and complex mixtures can be used as samples.As one of the interacting partners is immobilized on the sensor'ssurface and the second compound is injected into the flow system, themeasurement of equilibrium and transition state thermodynamics ispossible, as the FIA (Flow Injection Analysis) system allows for and canseparately monitor the complex association and dissociation phase.

With this technology the calculation of the thermodynamic parameters

-   -   free standard binding enthalpy ΔG^(∘),    -   standard binding enthalpy ΔH^(∘),    -   standard binding entropy ΔS^(∘),        and of the transition state parameters    -   free standard association enthalpy ΔG^(∘)‡ass,    -   standard association enthalpy ΔH^(∘)‡ass,    -   standard association entropy ΔS^(∘)‡ass,    -   activation energy Eaass,    -   free standard dissociation energy ΔG^(∘)‡diss,    -   standard dissociation enthalpy ΔH^(∘)‡diss,    -   standard dissociation entropy ΔS^(∘)‡diss, and    -   dissociation energy Eadiss        is possible. In case of using the non-linear van't Hoff equation        also a ΔC_(p) value can be determined.

Generally an SPR-based kinetic antibody screening (see e.g. Steukers,M., et al., J. Immunol. Methods 310 (2006) 126-135; Rich, R. L., et al.,Anal. Biochem. 361 (2007) 1-6) is succeeded by a second step of higherresolution thermodynamic SPR analyses.

Antibody producing cell are cultured and the produced cell culturesupernatants are subjected to, in one embodiment high throughput,analyses, in which the temperature-dependent kinetic data is generatedin order to calculate the velocity factor and transition state (TS)thermodynamic properties. The selection of the antibody according to themethod as reported herein is done based on its thermodynamic behavior.

A high affinity antibody is characterized by a temperature-dependentacceleration of the antigen complex association rate constant ka [1/Ms]and a remaining or decelerated antigen complex dissociation rateconstant kd [1/s]. Surprisingly, such an antibody is characterized by anantigen interaction mechanism, which shows a large entropy-change in thebinding equilibrium. Therefore, a risk assessment must take place byother means than calorimetry to determine from which effect the entropiccontribution originates. From the complex association phase, which isrisky due to promiscuitive antigen binding, or from the complexdissociation phase, which just indicates a complex antigen interaction,comprising a potentially induced conformational change of the antigen.In one embodiment the entropic contribution comes from theantibody-antigen complex dissociation step, wherein a large positive ora large negative change of the dissociation entropy ΔS^(∘)‡diss takesplace.

A high affinity antibody may also have a thermodynamic anomalyoriginating from the antigen dissociation phase, where the dissociationrate constant kd [1/s] surprisingly and unexpectedly decreases withincreasing temperature. Such an antibody is characterized bythermodynamic parameters such as i) a dissociation phase showing anegative or nearby zero dissociation activation energy Eadiss [kJ/mol],ii) a negative dissociation enthalpy ΔH^(∘)‡diss [kJ/mol], and iii) alarge negative dissociation entropy ΔS^(∘)‡diss [kJ/mol]. It has to bepointed out that this is a completely theoretical treatment of thiseffect and shall not be construed as limitation. Thus, in one embodimentthe dissociation activation energy Eadiss, the dissociation enthalpyΔH^(∘)‡diss, and the dissociation entropy ΔS^(∘)‡diss are determined andan antibody is selected that has i) a negative or nearby zerodissociation activation energy Eadiss [kJ/mol], ii) a negativedissociation enthalpy ΔH^(∘)‡diss [kJ/mol], and iii) a large negativedissociation entropy ΔS^(∘)‡diss [kJ/mol].

Thus, a method as reported herein allows for the selection of anantibody from a multitude of high affinity antibodies, based on thevelocity factor and the ΔS^(∘)‡ass value.

Single cell deposited clones can be cultivated prior to screening as inone embodiment in 100 ml spinner culture flasks using RPMI 1640 medium.In another embodiment the antibodies are purified from the supernatantby Protein A Sepharose™ column chromatography prior to the determinationof the temperature-dependent kinetic data, i.e. the thermodynamicscreening. In one embodiment the system buffer is HBS-EP for thethermodynamic screening. In another embodiment the sample buffer issupplemented with 1 mg/ml carboxymethyldextrane to reduce unspecificsensor matrix effects.

Most publications using SPR-based measurements don't use antibodycapture systems as sensor surface presentation technology. Usually theantibody or fragments thereof are covalently immobilized on the sensor.This technology can't be used in a high throughput format, since thesurface is not suitable for multi purpose antibody presentation, but itis technically limited by the number of sensors being immobilized withligands.

In order to perform a thermodynamic screening a species specific capturesystem with appropriate temperature-dependent secondary antibody complexstability can be established. The biosensor has to be calibrated by anoptimization procedure to determine the binding characteristics ofantibodies with varying epitope specificities in a high throughputformat. The thermodynamic screening provides a temperature-dependent setof data (see FIG. 3). At lower temperatures less response is observed,since the capture system's association rate is reduced (see Example 5).At higher temperatures the association rate accelerates.

Further has it been found that at temperatures below 13° C. the capturesystem's association kinetics are too slow for a sufficient antibodyresponse. Below 13° C. and above 37° C., the antibody's antigen bindingkinetics determination provide for non-linearizable data according tothe van't Hoff, Eyring and Arrhenius equations.

In one embodiment the thermodynamic screening is performed attemperatures of 13° C. or 17° C. and 37° C. It has been found that inthis temperature range a simple calculation of the thermodynamicequilibrium data according to the linear form of the van't Hoff equationand a simple calculation of transition state thermodynamics according tothe linear Eyring and linear Arrhenius equations is possible (Wear, M.A., et al., Anal. Biochem. 359 (2006) 285-287; see also FIG. 5). In oneembodiment all measurements are performed under the same conditions inorder to make high-throughput-screening (HTS) amenable.

For the calculation the following formulas can be used:

-   -   a) Arrhenius equation:        k=A*e ^((−Ea/R*T))  (I)    -   b) van't Hoff calculations:        ΔG ^(∘) =ΔH ^(∘) −T*ΔS ^(∘)  (II)        ΔG ^(∘) =−R*T*ln K _(D)  (III)        ln K _(D)=−1/T*(ΔH ^(∘) /R)/slope−(ΔS ^(∘) /R)/intercept  (IV)        R*T*ln K _(D) =ΔH ^(∘) _(T0) −T*ΔS ^(∘) _(T0) +ΔC ^(∘) _(p)(T−T        ₀)−T*ΔC _(p) ^(∘)ln(T/T ₀)  (V)    -   c) Eyring association phase:        ka=(k _(b) *T/h)*e ^((−ΔG) ^(o) ^(‡/R*T))  (VI)        ln ka/T=−1/T*(ΔH ^(∘) ‡/R)/slope+(ΔS ^(∘) ‡*R+ln k _(b)        /h)/intercept  (VII)        ka=A*e ^(−Ea/R*T)  (VIII)        ln ka=ln A/intercept−(1/T*Ea/R)/slope  (IX)    -   d) Eyring dissociation phase:        kd=(k _(b) *T/h)*e ^((−ΔG) ^(o) ^(‡/R*T))  (X)        ln kd/T=−1/T*(ΔH ^(∘) ‡/R)/slope+(ΔS ^(∘) ‡/R+ln k _(b)        /h)/intercept  (XI)        k _(d) =A*e ^(−Ea/R*T)  (XII)        ln kd=ln A/intercept−(1/T*Ea/R)/slope  (XIII)    -   with        -   ΔH^(∘)—standard binding enthalpy,        -   ΔS^(∘)—standard binding entropy,        -   ΔG^(∘)—free standard binding enthalpy,        -   T*ΔS^(∘)—entropic term,        -   ΔH^(∘)‡ass—standard association binding enthalpy,        -   ΔS^(∘)‡ass—standard association binding entropy,        -   ΔG^(∘)‡ass—standard association free binding enthalpy,        -   Eaass—Arrhenius Parameter for the association,        -   ΔH^(∘)‡diss—standard dissociation binding enthalpy,        -   ΔS^(∘)‡diss—standard dissociation binding entropy,        -   ΔG^(∘)‡diss—standard dissociation free binding entropy,        -   Eadiss—Arrhenius Parameter for the dissociation,        -   K_(D)—affinity constant,        -   ka—association rate constant,        -   k_(b)—Boltzmann Constant=(1.3806503×10⁻²³ m² kg s⁻² K⁻¹),        -   kd—dissociation rate constant,        -   h—Planck constant,        -   C_(p)—molar heat capacity.

The temperature dependency of the free binding enthalpy ΔG^(∘)can becalculated for each temperature in the range from 13° C. to 37° C. withthe formula ΔG^(∘)=−R*T*ln K_(D). If the value is constant, the linearform of the van't Hoff equation can be used. If ΔG^(∘)changes thenon-linear form is preferred.

The data obtained in the thermodynamic screening can be visualized in adouble logarithmic plot as depicted in FIG. 6 a) wherein the kineticrate constants (k_(on)) ka [1/Ms] and (k_(off)) kd [1/s] are denoted onthe X- and Y-axis, respectively. Isometric lines (solid lines) indicateareas of the same affinities, which are plotted in bold at the rightside of the diagram. Since the ratio of kd/ka provides for theequilibrium constant K_(D) [M], each data point is equivalent to anaffinity at a respective temperature. The arrow above symbolizes thetemperature gradient in steps of +4° C. starting at 13° C. or 17° C.,respectively, and ending at 37° C. Temperature-dependent affinity trendsof each antibody are connected by a line.

Three exemplary affinity trends are shown in FIG. 6 b). In this rate mapthe K_(D)s in steps of +4° C. of three example antibodies are shown,whereof one is an antibody with increasing affinity with increasingtemperature, one is an antibody with constant affinity with increasingtemperature, and one is an antibody with decreasing affinity withincreasing temperature. Most antibodies show an affinity loss due tolacking antigen complex stability (like those represented by the squarein FIG. 6 b)). The affinity remains constant when k_(on) and k_(off)increase (circles). A therapeutic or diagnostic antibody preferablyshows an increase in k_(on) and k_(off) with increasing temperature orshows a k_(on) acceleration and a k_(off) deceleration. Likewise theaffinity increases and the complex gains stability at highertemperatures (filled circles).

The monitoring of the temperature-dependent kinetics as shown in FIG. 6is the basis for the selection of antibodies withtemperature-independent or temperature-increasing antigen complexstability.

FIG. 7 exemplarily shows two different temperature-dependent kinetics oftwo antibody forms 1 and 2, which can be obtained in a humanizationprocess. In such a process different combinations of human germlineheavy and light chain based sequences can be used to replace murineparental antibody framework sequences. The aim is to select humanizedmonoclonal antibodies (mAbs) with, preferably, unchanged thermodynamics,specificities and stabilities, when compared to the parental antibody.It can be seen, that antibody 2, in contrast to antibody 1, stronglyaccelerates its association rate constant ka in the given temperaturegradient.

Both antibodies bind the antigen at 37° C. with similar affinity, sothat affinity is no suitable selection parameter to discriminate theantibodies.

Since both antibodies strongly differ in their entropic contributions, amuch better additional selection parameter is the ΔS^(∘)‡ass. Forexample, the humanized antibody form 1 shows ΔS^(∘)‡ass=70+/−15 J/mol*K(Eyring equation, R²=0.9601) and humanized antibody form 2 shows abinding entropy of ΔS^(∘)‡ass=350+/−70 J/mol*K (Eyring equation,R²=0.9530). The higher binding entropy of humanized antibody form 2 isreflected by a strong acceleration of the association rate constant kain the temperature gradient of from 13° C. to 37° C.

It has been found that the acceleration of the antibody-antigen complexformation by a temperature increase, respectively the increase of theassociation rate constant ka at elevated temperature, correlates withthe antibody-antigen association phase entropy ΔS^(∘)‡ass.

The antigen-binding interactions of 28 antibodies were thermodynamicallyquantified. The association phase entropy ΔS^(∘)‡ass and the velocityfactor VF (ka(37° C.)/ka(13° C.)) were calculated (see Table 1).

TABLE 1 ΔS°‡ass of 28 antibodies calculated according to the Eyringequation; all values show R² > 95%. SE: error of the calculation. VF:Velocity Factor. ΔS°‡ass SE antibody antigen VF [J/mol * K] [J/mol * K]antibody 3  4 2 −85 7 antibody 4  5 2 −53 5.1 antibody 5  3 2 −77 5.4antibody 6  4 2 −73 6.9 antibody 7  4 2 −83 1.2 antibody 8  2 3 −18 3.5antibody 9  2 3 −10 5.8 antibody 10 5 3 −66 14 antibody 11 1 2 −35 4.1antibody 12 5 2 −38 13 antibody 13 1 5 −14 1.3 antibody 14 2 5 9 6.6antibody 15 1 5 −5.3 5.6 antibody 16 1 6 71 23 antibody 17 2 8 77 15antibody 18 2 9 80 16 antibody 19 2 12 110 29 antibody 20 2 21 160 41antibody 21 2 74 350 70 antibody 22 2 137 370 78 antibody 23 2 4 6 5.5antibody 24 2 4 3.6 4.2 antibody 25 2 5 26 9.2 antibody 26 6 5 38 13antibody 27 2 5 24 6.3 antibody 28 2 6 34 12 antibody 29 2 6 28 12antibody 30 6 7 62 14 antibody 31 2 8 77 15

Table 1 shows data of 28 different murine, human chimeric and humanizedmurine antibodies, Fab and Fab′2 fragments from murine or human origin,binding to six different antigens. The antigens are all proteinogenous,conformational antigens, differing in their molecular weight. TheVelocity Factor VF correlates with ΔS^(∘)‡ass, whereby small values ofVF correlate with negative or small values for ΔS^(∘)‡ass. It has beenfound that a negative ΔS^(∘)‡ass indicates an enthalpy-drivenantibody-antigen interaction, a positive ΔS^(∘)‡ass values indicateentropic/enthalpic interactions and three digit ΔS^(∘)‡ass values standfor fully entropy-driven interactions. Thus, herein is reported a methodfor selecting a specifically binding antibody comprising the followingsteps

-   -   a) providing a multitude of antibodies,    -   b) determining the association rate constant of each antibody to        its antigen at 37° C. and the association rate constant of each        antibody to its antigen at 13° C.,    -   c) calculating the ratio of the association rate constant at        37° C. to the association rate constant at 13° C.,    -   d) selecting a specifically binding antibody by selecting an        antibody with a ratio of 10 or less and a ΔS^(∘)‡ass value of        100 J/mol*K or less.

In one embodiment the selecting is of an antibody with a ΔS^(∘)‡assvalue of 50 J/mol*K or less.

The higher ΔS^(∘)‡ass the higher is the probability of promiscuitiveantibody binding.

Small or negative ΔS^(∘)‡ass correlates to specificity and monospecificbinding.

It has been found that it is not necessary to determine associationphase thermodynamics by linearizing the data according to the Eyring andArrhenius equations, where it is the task to produce linearizable highquality data in order to calculate the parameters with acceptableerrors. Using the methods as reported herein it is sufficient todetermine kinetic data at only two temperatures, i.e. at 13° C. and at37° C.

Based on the experimental data the values ka(37° C.) and ka(13° C.) aredetermined by using an appropriate kinetic model. Thereafter theVelocity Factor (VF)=ka(37° C.)/ka(13° C.) is calculated and ΔS^(∘)‡assis assessed according to the characteristic curve presented in FIG. 8.

Double digit VF-values correlate with three digit ΔS^(∘)‡ass values andindicate a higher risk of promiscuitive binding. The data in FIG. 8 weremodeled by an exponential association functiony=y0+A1*(1−exp(−x/t1))+A2*(1−exp(−x/t2)). According to the graph of theexponential equation, the risk to select an unwanted entropy-drivenbinder can be minimized. ΔS^(∘)‡ass is estimated by the surrogateparameter VF. In contrast to ΔS^(∘)‡ass, VF is an instrumentally easy toaccess parameter.

FIG. 8 shows that ΔS^(∘)‡ass reaches a plateau value at the equationparameter A1=399.74088+/−36.60886 J/mol*K.

The more relevant data is characterized by small VF values of less than10 and show smaller standard deviations of ΔS^(∘)‡ass.

The fitting parameter A2 (A2=198.50729+/−29.933 J/mol*K) is ΔS^(∘)‡assat the inflexion of the curve.

Antibodies with ΔS^(∘)‡ass of more than A2 (ΔS^(∘)‡ass>A2) are critical,i.e. not well suited, for further processing efforts. FIG. 8 indicates,that most of the antibodies analyzed show VF values of less than 10(<10). These antibodies can be diagnostically or pharmaceutically used.

Antibodies with ΔS^(∘)‡ass of more than A2, which correlates to a VF ofmore than 50 show e.g. cross reactivity to further antigens.

Thus, herein is reported a method for selecting a cross-reactiveantibody comprising the following steps

-   -   a) providing a multitude of antibodies,    -   b) determining the association rate constant of each antibody to        its antigen at 37° C. and the association rate constant of each        antibody to its antigen at 13° C.,    -   c) calculating the ratio of the association rate constant at        37° C. to the association rate constant at 13° C.,    -   d) selecting a cross-reactive antibody by selecting an antibody        with a ratio of 50 or more and a ΔS^(∘)‡ass value of 200 J/mol*K        or more.

In one embodiment the selecting is of an antibody with a ΔS^(∘)‡assvalue of 300 J/mol*K or more.

FIG. 8 can be segmented into four corridors (see FIG. 9). Factors0<VF<20 indicate a selected screening result. Antibodies in segment 3show VF factors of more than 20 (VF>20) and are not selected due totheir increased ΔS^(∘)‡ass values. These antibodies are suspicious topromiscuitive antigen binding and require more detailed analyses toclarify the target binding mechanism.

The classification according to FIG. 9 can be used to realize highthroughput ΔS^(∘)‡ass based screenings of large antibody populations.For example in the humanization process of antibodies, where the optimalhuman heavy and light chain compositions are systematically tested fortheir optimal performance, similar to the parental antibody, the methodas reported herein can be used.

The antibodies 8, 9, 17, 18, 20, 21, 22, and 31 as comprised in Table 1correspond to different stages of a humanization process. From the VFvalues the process of humanization can be followed. The parental murineantibody 8 shows a low VF value and a negative ΔS^(∘)‡ass. The chimericantibody 9 shows still unchanged VF as well as ΔS^(∘)‡ass. The fullyhumanized antibodies, which are of different VL/VH chain sequencecombinations, show discrepancies of VF and therefore of ΔS^(∘)‡ass.Finally selected was antibody 31, which shows the lowest VF as well asΔS^(∘)‡ass, whereas antibody 21 and antibody 20 were not selected due tothermodynamic instability, lower functionality in cell culture assays,and/or promiscuitive antigen binding.

Table 1 indicates that the diagram shown in FIG. 9 can also be used forantibody fragments.

The method as reported herein can also be used for obtaining antibodiesor antibody producing cells e.g. directly originating from cellmixtures, in one embodiment hybridoma or B-cells, or in anotherembodiment from a single deposited hybridoma or B-cell, whereinantibodies of potentially inhomogeneous maturation status are excluded,because it is of importance to identify fully mature antibodies withenthalpic binding behavior.

The method as reported herein can be used in high throughput format.Generally, ΔS^(∘)‡ass values are calculated from time-consumingmeasurements by using a wide-spanning temperature-range and as many dataspots as possible in order to get a good linear correlation to calculatetransition state parameters according to the Eyring equation withR²>95%.

In the methods as reported herein by using only two temperatures, 13° C.and 37° C., and the reduced kinetic “2 over 2” model precise velocityfactors for the association rate can be obtained. This is essential forhigh throughput ΔS^(∘)‡ass measurements. When combining the highthroughput kinetic screening for the identification of highly stableantigen complex forming antibodies with the high throughputdetermination of the VF factors, best in class antibodies can beselected.

For example, when the VF values in Table 2 are referenced to theΔS^(∘)‡ass/VF diagram it can be seen, that the monoclonal antibodydenoted as 1F8 does not show elevated association phase entropy. Thishas been confirmed also in a different experimental format. The mean VFvalue of all 16 buffer conditions is 4. The VF value in the referenceTable 1 is 4 under similar buffer conditions. This example givesevidence for the applicability of high throughput ΔS^(∘)‡ass analyses byusing the VF factors as a ΔS^(∘)‡ass surrogate.

TABLE 2 Matrix showing the Velocity Factors (VF = ka (37° C.)/ka (13°C.)) of the antibody 1F8/antigen interactions for 16 different bufferconditions. All interactions were measured in PBS with varying KClconcentrations and different pH values. Antibody 1F8 would thereforepopulate segment 1 of FIG. 9. 1F8 pH ↓ KCl [mM] → 2.7 54 162 324 6.8 2 46 4 7.0 4 3 4 3 7.4 4 5 4 4 7.8 4 4 4 5

The antibody selected with a method as reported herein can be producedrecombinantly. Methods for recombinant production of antibodies areknown in the state of the art and comprise protein expression inprokaryotic and eukaryotic cells with subsequent isolation of theantibody and usually purification to a pharmaceutically acceptablepurity. For the expression of the antibodies as aforementioned in a hostcell, nucleic acids encoding the respective light and heavy chains canbe inserted into expression vectors by standard methods. Expression canbe performed in appropriate prokaryotic or eukaryotic host cells likeCHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6(R)cells, yeast, or E. coli cells, and the antibody can be recovered fromthe cells (supernatant or cells after lysis). General methods forrecombinant production of antibodies are well-known in the state of theart and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-160; Werner, R. G., Drug Res. 48 (1998) 870-880.

The term “host cell” as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodiesaccording to the current invention. In one embodiment HEK293 cells andCHO cells are used as host cells. As used herein, the expressions“cell,” “cell line,” and “cell culture” are used interchangeably and allsuch designations include progeny. Thus, the words “transformants” and“transformed cells” include the primary subject cell and culturesderived there from without regard for the number of transfers. It isalso understood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included.

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids Res. 30 (2002) E9. Cloning of variabledomains is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89(1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods 204(1997) 77-87. A transient expression system (HEK 293) is reported bySchlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.

The term “specifically binding” denotes the binding of an antibody orFab fragment with a dissociation constant (=K_(Diss)) of at least 10⁻⁸mol/l or less, i.e. 10⁻⁹ mol/l, or in the range of 10⁻⁸ to 10⁻¹³ mol/1.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 Illustration of the Binding Late (BL) and Stability Late (SL)data of exemplary antibodies.

FIG. 2 Binding Late/Complex Stability plot of 549 hybridoma primarycultures: the encircled data spot shows sufficient antigen responsesignal and 100% complex stability, whereas the enframed data spot showsno sufficient antigen response.

FIG. 3 Secondary antibody response of the <IgGFCγM>R antibody capturesystem versus the analyte monoclonal antibody at 25 nM, 50 nM, 75 nM and100 nM and at increasing temperatures.

FIGS. 4a-b Exemplary concentration-dependent sensogram of thetemperature-dependent antibody-antigen interactions: (a) of antibody MD1.1. The kinetics were measured in HBS-EP pH 7.4 at 25° C., 3 min.association time, 5 min. dissociation time, fitting according toLangmuir model; and (b) of antibody M 9.3.1. The kinetics were measuredin HBS-EP pH 7.4 at 25° C., 3 min. association time, 15 min.dissociation time, fitting according to a Langmuir 1.1. model.

FIGS. 5a-c Calculation of thermodynamic parameters according to thelinear equations of: (a) van't Hoff, (b) Eyring and (c) Arrhenius.Exemplary plots are shown for antibody MD1.1.

FIGS. 6a-b a) Double logarithmic plot of the temperature-dependentcharacteristics of 34 exemplary antibodies; and b) Double logarithmicplot of the temperature-dependent characteristics of three exemplaryantibodies: filled circles—antibody with increasing affinity withincreasing temperature, open circles—antibody with constant affinitywith increasing temperature, squares—antibody with decreasing affinitywith increasing temperature.

FIG. 7 Rate map with double logarithmical plotting of the antigendissociation constant kd [1/s] versus the antigen association rateconstant ka [1/Ms]; isometric lines indicate areas of constant affinity;the temperature-dependent kinetics of two humanized antibodies, antibody1 (filled triangles) and antibody 2 (hollow stars) are plotted in thetemperature gradient 13° C. to 37° C. in steps of +4° C., beginning atslower association rates at the left of the graphic.

FIG. 8 Data of Table 1. ΔS^(∘)‡ass is plotted versus VF. The data wasfitted with an exponential association model according to the equationy=y0+A1*(1−exp(−x/t1))+A2*(1−exp(−x/t2)); Y0=−185.05789+/−36.83135;A1=399.74088+/−36.60886; t1=41.93317+/−9.834; A2=198.50729+/−29.933;t2=3.13298+/−1.32572; R²=0.97575. The dashed line at VF=10 indicates,that most of the suitable interactions show VF<10.

FIG. 9 Segmentation of the plot of FIG. 8 in four corridors by shortdotted lines. The segments are numbered 1, 2, 3 and 4.

EXAMPLE 1 Immunization of Mice

Balb/c mice 8-12 weeks old were subjected to intraperitonealimmunization with 100 μg of the antigen formulated as a KLH (keyholelimpet haemocyanine) fusion in complete Freud's adjuvant.

The immunization was performed 4 times: initial boost, 6 weeks, 10 weeksand 14 weeks after the initial boost. The second and third immunizationwas done using incomplete Freud's adjuvant. The final boost was donei.V. using 100 μg antigen three days before the hybridoma fusion tookplace. The production of hybridoma primary cultures was done accordingto Köhler and Milstein (Kohler, G., et al., Nature 256 (1975) 495-497).The hybridomas were isolated in 96-well micro titer plates (MTPs) bylimited dilution and were screened for antigen binding by ELISA methodsaccording to the manufacturer's manual. Primary hybridoma cell cultures,which showed a positive color formation upon antigen binding in ELISA,were transferred into the kinetic screening process.

EXAMPLE 2 Preparation of the CM5 Sensor Chip

The BIAcore A100 system under the control of the Software V.1.1 wasprepared like follows: A BIAcore CM5 sensor (series S) was mounted intothe system and was hydrodynamically addressed according to themanufacturer's recommendations.

In case of analyzing a murine antibody, the polyclonal rabbit anti-IgGantibody <IgGFCγM>R (Jackson ImmunoResearch Laboratories Inc.) wasimmobilized on the flow cells via EDC/NHS chemistry according to themanufacturer's instructions.

In case of using human chimeric or fully humanized antibodies, thepolyclonal goat antibody pAb<h-IgG, Fcg-Frag>G-IgG(IS) (JacksonImmunoResearch Laboratories Inc.) was immobilized on all flow cells viaEDC/NHS chemistry according to the manufacturer's instructions.

In case of using murine IgG Fab or Fab′2 fragments, the polyclonal goatantibody <MFab>G-IgG(IS) (Bethyl L. Cat. No. A90-100A-5 v. 9.8.2000) wasimmobilized on all flow cells via EDC/NHS chemistry according to themanufacturer's instructions.

In case of using human or humanized IgG Fab or Fab′2 fragments, thepolyclonal goat antibody <huFab′2>G-IgG (Jackson Immuno ResearchLaboratories Inc.) was immobilized on all flow cells via EDC/NHSchemistry according to the manufacturer's instructions.

EXAMPLE 3 Kinetic Screening of Primary Hybridoma Culture Supernatants

Hybridoma culture supernatants from different immunization campaignsconducted according to Example 1 were processed as outlined below.

The spots 2 and 4 of a sensor chip obtained according to Example 2 wereused as a reference (1-2, 5-4). In order to capture antibody on thesensor surface hybridoma culture supernatants were diluted 1:5 withrunning buffer HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05%P20, BIAcore) and were injected at 30 μl/min. for 1 min. Subsequently,the respective antigen was injected at 30 μl/min. for 2 to 3 min.association time. The dissociation phase was monitored for 5 to 15 min.Finally the surface was regenerated with a 2 min. injection of 100 mMphosphoric acid.

The sensor was preconditioned by repeated cycles of antibody capturingand regeneration.

For the selection of primary hybridomas the following procedure wasused: A Binding Late (BL) reference point was set shortly before theantigen's injection ended. A Stability Late (SL) reference point was setshortly before the end of the complex dissociation phase. The BL and SLdata were graphically visualized (FIG. 1). The data was used tocalculate the antigen complex stability using formula (XIV):(1−[BL(RU)−SL(RU)/BL(RU)])  (XIV)(see FIG. 2). E.g. the encircled data spots show sufficient antigenresponse signal and 100% complex stability, whereas the enframed dataspot shows no sufficient antigen response.

Thus, the top 10% hybridomas according to antigen response signal andcomplex stability have been selected.

EXAMPLE 4 Hybridoma Cloning and Antibody Production

Antibody producing hybridoma primary cultures selected according toExample 3 were subcloned using the cell sorter FACSAria (BectonDickinson) under the control software V4.1.2. The deposited singleclones were incubated under suitable conditions for furtherproliferation in 24 well plates and were subsequently transferred to thethermodynamic screening process according to Example 5 after havingdetermined the antibody concentration in solution using ELISA methodsaccording to the instruction of the manufacturer.

EXAMPLE 5 Thermodynamic Screening

Secreted antibodies were characterized by a thermodynamic screeningemploying the determination of the temperature-dependent kinetics inorder to determine the antigen-antibody complex thermostability and inorder to calculate thermodynamic properties.

A CM5 sensor series S was mounted into the BIAcore T100 System drivenunder the control software V1.1.1 and preconditioned by 1 min. injectionat 100 μl/min. of a mixture comprising 0.1% SDS, 50 mM NaOH, 10 mM HCland 100 mM H₃PO₄.

In case of analyzing a murine antibody, the polyclonal rabbit anti-IgGantibody <IgGFCgammaM>R (Jackson ImmunoResearch Laboratories Inc.) wasimmobilized on the flow cells via EDC/NHS chemistry according to themanufacturer's instructions.

In case of using human chimeric or fully humanized antibodies, thepolyclonal goat antibody pAb<h-IgG, Fcg-Frag>G-IgG(IS) (JacksonImmunoResearch Laboratories Inc.) was immobilized on all flow cells viaEDC/NHS chemistry according to the manufacturer's instructions.

In case of using murine IgG Fab or Fab′2 fragments, the polyclonal goatantibody <MFab>G-IgG(IS) (Bethyl L. Cat. No. A90-100A-5 v. 9.8.2000) wasimmobilized on all flow cells via EDC/NHS chemistry according to themanufacturer's instructions.

In case of using human or humanized IgG Fab or Fab′2 fragments, thepolyclonal goat antibody <huFab′2>G-IgG (Jackson Immuno ResearchLaboratories Inc.) was immobilized on all flow cells via EDC/NHSchemistry according to the manufacturer's instructions.

The concentration values of the reference antibody were adjusted inorder to achieve similar secondary antibody response levels at differenttemperatures.

Kinetic measurements at different temperatures were performed at 20μl/min., the flow rate was 30 μl/min., 50 μl/min., 100 μl/min.,respectively. The sample injection of the antigen was done for 30 sec.,90 sec., 180 sec., respectively, or other suitable injection times inorder to achieve ligand saturation or entry into the binding equilibriumduring the complex association phase (see FIG. 4 a)). The dissociationrate was monitored first for up to 300 sec. and further for 15 min. (seeFIG. 4 b)). The antigen injections were repeated in differentconcentration steps of at least five concentrations. As control oneconcentration step was analyzed twice to control the reproducibility ofthe assay. Flow cell 1 served as a reference. A buffer injection wasused instead of an antigen injection to double reference the data bybuffer signal subtraction. The capture system was regenerated using 100mM H₃PO₄ by a 2 min. injection at 100 μl/min. The regeneration procedurewas optimized to guarantee quantitative surface regeneration also at 13°C., 17° C. and 21° C. At these temperatures the regeneration solutionwas injected three times whereas at 25° C., 29° C., 33° C. and 37° C.the regeneration solution was injected one time.

The data obtained was evaluated according to a 1:1 binary Langmuirinteraction model in order to calculate the association rate constant ka[1/Ms], the dissociation rate constant kd [1/s] and the resultingaffinity constant K_(D) [M] at different temperatures. Thermodynamicequilibrium data was calculated according to the linear form of theVan't Hoff equation. Transition State thermodynamics were calculatedaccording to the Eyring and Arrhenius equations using e.g. the BIAcoreT100 evaluation software V.1.1.1 or the program Origin 7SRI v. 7.0300.

EXAMPLE 6 Example for the Effect of the Adjustment to HomogeneousRU_(MAX) Values

A BIAcore T100 device was mounted with a CM5 series-S BIAcore sensor,and was immobilized with 6000 RU<IgGFCyM>R (Jackson ImmunoResearchLaboratories Inc., USA) on each flow cell according to themanufacturer's instructions. The non-optimized experiment used 40 nMcapture antibody at 20 μl/min., in HBS-EP buffer (0.05% P20). The samplebuffer was the system buffer, supplemented with 1 mg/ml CMD(carboxymethyldextrane).

The antigen was injected after the capturing of the secondary antibodyin six concentration steps of 1.2 nM, 4 nM, 11 nM, 33 nM, 100 nM and 300nM, whereby 11 nM were used as a double control and 0 nM were used asreference. The antigen was injected at 100 μl/min. for 2 min.association and 5 min. dissociation, followed by a HBS-EP wash of 15min. at 30 μl/min. and a regeneration with 10 mM glycine pH 1.7 at 3μl/min. for 3 min. Concentration-dependent measurements were done at 4°C., 11° C., 18° C., 25° C., 32° C., and 40° C.

The optimized system was used like described above, but with theexceptions that the antibody to be captured was injected for 3 min.association time at different concentration steps of 100 nM at 15° C.,80 nM at 20° C., 60 nM at 25° C., 50 nM at 30° C., 40 nM at 35° C. and40 nM at 40° C.

Finally kinetics and thermodynamics were determined using the BIAcoreevaluation software.

EXAMPLE 7 Determination and Calculation of the Velocity Factor

A CM5 sensor series S was mounted into the BIAcore T100 System.

In case of using murine antibodies, the polyclonal rabbit IgG antibody<IgGFCyM>R (Jackson ImmunoResearch Laboratories Inc.) was immobilized onthe flow cells via EDC/NHS chemistry according to the manufacturer'sinstructions.

In case of using human chimeric or fully humanized antibodies, thepolyclonal goat antibody pAb<h-IgG, Fcg-Frag>G-IgG(IS) (JacksonImmunoResearch Laboratories Inc.) was immobilized on all flow cells viaEDC/NHS chemistry according to the manufacturer's instructions.

In case of using murine IgG Fab or Fab′2 fragments, the polyclonal goatantibody <MFab>G-IgG(IS) (Bethyl L. Cat. No. A90-100A-5 v. 9.8.2000) wasimmobilized on all flow cells via EDC/NHS chemistry according to themanufacturer's instructions.

In case of using human or humanized IgG Fab or Fab′2 fragments, thepolyclonal goat antibody <huFab′2>G-IgG (Jackson Immuno ResearchLaboratories Inc.) was immobilized on all flow cells via EDC/NHSchemistry according to the manufacturer's instructions.

The sample buffer was the system buffer supplemented with 1 mg/mlcarboxymethyldextrane to reduce unspecific sensor matrix effects.Kinetic measurements in the temperature gradient 13° C. to 37° C. wereperformed at 100 μl/min.

The analyte injections of recombinant synthetic human full lengthantigen 1, recombinant human antigen 2 (10 kDa), recombinant humanantigen 3 human FC-chimera (R&D Systems, 160 kDa), recombinant humanantigen 4 (29 kDa), or recombinant human antigen 5 (72 kDa) were donefor 180 sec. The dissociation rate was monitored for up to 900 sec. Theantigen injections were repeated in different concentration steps of atleast five concentrations. As a control one concentration step wasanalyzed twice to control the reproducibility of the assay. Flow cell 1was used as reference. A blank buffer injection was used instead of anantigen injection to double reference the data by buffer signalsubtraction.

Prior to each assay, homogenous RU_(MAX) values in the temperature range13° C.-37° C. were adjusted by titration experiments with the respectiveantibodies to be presented on the sensor surface (see section in whichthe temperature-dependent titration experiments are described indetail). The kinetics were measured in the temperature gradient 13° C.,17° C., 21° C., 25° C., 29° C., 33° C. and 37° C. The capture systemswere regenerated using a buffer wash with HBS-ET buffer at 30 μl/min.for 15 sec., prior to regeneration with 10 mM glycine pH 1.5 at 30μl/min. for 15 sec., followed by a 1 min. injection and a 30 sec.injection of 10 mM glycine at pH 1.7.

The data obtained was evaluated according to a 1:1 binary Langmuirinteraction model in order to calculate the association rate constantska [1/Ms], the dissociation rate constants kd [1/s] and the resultingaffinity constants K_(D) [M] at the respective temperatures.Thermodynamic equilibrium data was calculated according to the linearand non linear form of the Van't Hoff equation. Transition Statethermodynamics were calculated according to the Eyring and Arrheniusequations using e.g. the BIAcore T100 evaluation software V.1.1.1.Graphic evaluation was done using Origin 7SRI v. 7.0300.

The Velocity Factor (VF) was calculated as the quotient of the antigencomplex association rates ka (1/Ms) at 37° C. and 13° C. The exponentialassociation fitting curve y=y0+A1*(1−exp(−x/t1))+A2*(1−exp(−x/t2)) wasused.

EXAMPLE 8 High Throughput Velocity Factor Analysis

A CM5 sensor series S was mounted into the BIAcore A100 System and thedetection spots were hydrodynamically addressed according to themanufacturer's instructions.

The polyclonal rabbit IgG antibody <IgGFCyM>R (Jackson ImmunoResearchLaboratories Inc.) was immobilized at 4 kRU on the detection spots 1 and5 in each flow cell. 800 RU<IgGFCyM>R were immobilized on spots 2 and 4in each flow cell. Coupling was done via EDC/NHS chemistry according tothe manufacturer's instructions. The sample buffer was the system buffersupplemented with 1 mg/ml carboxymethyldextrane to reduce unspecificsensor matrix effects.

The basic buffer system was PBS (phosphate buffered saline). The bufferwas adjusted to four different pH conditions: pH 6.8, pH 7.0, pH 7.4 andpH 7.8, and four different KCl concentrations: 2.7 mM, 54 mM, 162 mM and324 mM. Sixteen different sample buffer conditions were tested.

The mAbs to be captured were injected at 10 μl/min. for 1 min. atdifferent concentration steps between 60 nM at 37° C. and 240 nM at 13°C. to ensure homogenous RU_(MAX) values in the subsequent antigeninteraction measurements.

Kinetic measurements in the temperature gradient 13° C. to 37° C. wereperformed at 30 μl/min. The analyte injections of recombinant humanantigen 1-84 (9.4 kDa), were done for 180 sec. The dissociation rate wasmonitored for 600 sec. The antigen injections were repeated in twoconcentration steps at 60 nM and 240 nM. A blank buffer injection wasused instead of an antigen injection to double reference the data bybuffer signal subtraction.

The capture system was regenerated using a 15 sec. buffer wash withHBS-ET buffer at 30 μl/min., prior to regeneration with 10 mM glycine pH1.5 at 30 μl/min. for 90 sec., followed by 2×30 sec. injection of thesame buffer.

The kinetic data obtained was evaluated according to a “2 over 2”kinetic model, which uses two different ligand densities of the sensormatrix to calculate kinetics by just using two antigen concentrations.The association rate constants ka [1/Ms], the dissociation rateconstants kd [1/s] and the resulting affinity constants KD [M] at therespective temperatures were calculated using the BIAcore A100evaluation software 1.1.

Thermodynamic equilibrium data was calculated from the kinetic dataaccording to the linear form of the Van't Hoff equation. TransitionState thermodynamics were calculated according to the Eyring equationsusing Excel. Graphic evaluation was done using Origin 7SRI v. 7.0300.The Velocity Factor (VF) for the association phase was calculated as thequotient of the antigen complex association rates ka (1/Ms) at 37° C.and 13° C. The Velocity Factor for the dissociation rates werecalculated as the quotient of kd (1/s) at 13° C. and 37° C.

The invention claimed is:
 1. A method for obtaining an antibody withcross-reactivity for at least two antigens comprising the followingsteps: a) providing a multitude of antibodies, b) measuring theassociation rate constant of each antibody to an antigen at 37° C. andthe association rate constant of each antibody to the same antigen at13° C., c) calculating the ratio of the association rate constant at 37°C. to the association rate constant at 13° C., d) selecting an antibodywith a ratio of 50 or more and thereby obtaining the antibody withcross-reactivity for at least two antigens.